CN110923206A - Method for preparing antigen presenting cell - Google Patents
Method for preparing antigen presenting cell Download PDFInfo
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- CN110923206A CN110923206A CN201811103108.8A CN201811103108A CN110923206A CN 110923206 A CN110923206 A CN 110923206A CN 201811103108 A CN201811103108 A CN 201811103108A CN 110923206 A CN110923206 A CN 110923206A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
Abstract
The invention discloses a method for modulating antigen presenting cells, which is characterized in that under the conditions of in vitro and in the presence of nucleotide, antigen is added into the antigen presenting cells; the antigen-added antigen presenting cells are dendritic cells. The present invention utilizes the properties of antigen presenting cells to make it possible to manufacture a medicament for the treatment of cancer and infectious diseases; in addition, since the nucleotide has the property of activating antigen-presenting cells, it can act as an antigen-presenting cell activator; by using an antigen presenting cell activator, antigen presenting cells can be modulated in vitro; if administered to a patient, the condition of the patient can be improved by activating antigen presenting cells.
Description
Technical Field
The invention relates to the field of immunization, in particular to a method for modulating antigen presenting cells.
Background
In recent years, immunocytotherapy has been attracting attention as a method for treating diseases including cancer. The immune cell therapy is a therapeutic method in which immune cells of a patient are extracted and activated, and the activated immune cells are implanted into the patient to artificially enhance the immunity. Since immune cells specifically attack pathogenic cells and cells of the patient himself/herself who uses the immune cells, the immune cell therapy has fewer side effects than conventional anticancer agents. Vaccine therapy of dendritic cells (hereinafter abbreviated as DC) is one of immune cell therapies.
In order to apply the DC vaccine to cancer treatment, guiding an effective anti-tumor immune response of an individual can be achieved by improving the immune activation capability of the DC.
Therefore, it is an urgent problem to be solved by those skilled in the art to provide a method for improving the immune activation ability of DC.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing antigen-presenting cells, which improves immune response when providing antigen-presenting cells to a patient by allowing a single amount of nucleotides to coexist during preparation of the antigen-presenting cells; by utilizing the properties of the antigen-presenting cells prepared by the preparation method of the present invention, a pharmaceutical composition effective for treating cancer and infectious diseases is produced.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing antigen presenting cells, comprising adding an antigen to antigen presenting cells under in vitro conditions in the presence of nucleotides; the antigen presenting cells are dendritic cells; the nucleotide is ribonucleotide or nucleoside-phosphate; the nucleotide is uridylic acid or adenylic acid.
An antigen presenting cell is a cell (T cell, B cell, etc.) that can present an antigenic peptide in MHC class I and/or class II and direct a specific action of the antigen.
The antigen includes cancer or infectious antigen protein, cancer or infectious antigen peptide, and lysate of pathogenic cell (tumor tissue or tumor cell, infectious cell infected with virus, or disrupted product of pathogenic bacterium). In addition, the DNA and mRNA of the antigen can be found by transcription and translation.
Nucleotides may be added to the antigen presenting cell culture or suspension before or after the antigen presenting cell injection process (during installation).
The best effect is achieved when the concentration of nucleotide in the solution is 10-1000. mu.M.
Further, the antigen-specific Th1 cells were able to recognize the added antigen, and the antigen-added antigen-presenting cells had a stimulatory effect on Th1 cells.
Th1 cells are activated and stimulated by CD 4-positive helper T cells (Th cells) to produce interferon-gamma (hereinafter referred to as IFN-. gamma.) to enhance cellular immunity.
An antigen-specific Th1 cell is a Th1 cell in which an antigen presented by class II antigen presenting cells is recognized by a T cell receptor and activated.
Further, an antigen-presenting cell prepared by the method for preparing an antigen-presenting cell.
Methods for injecting antigen into antigen-presenting cells include co-culture and electroporation.
The co-culture method is a method of obtaining an antigen by the bulimia function of antigen-presenting cells. Antigen-presenting cells and antigen are suspended in the same vessel and cultured under common culture conditions for more than 2 hours to allow the antigen-presenting cells to enter the antigen.
The electroporation method is a method of introducing an antigen by utilizing the physical principle of breaking membranes of prokaryotic and eukaryotic cells when an electric pulse is applied to the cells. For example, when an antigen protein or the like is added to a cell culture solution, the antigen protein can be directly introduced into the cytoplasm from the pores of the expanded cell membrane by applying an electric pulse.
Antigen-specific Th1 cells (CD 4) were prepared by co-culture or electroporation to obtain antigen-specific antigen-presenting cells compared to antigen-presenting cells implanted with antigen alone+T cells) are better activated.
Further, a pharmaceutical composition comprising the antigen-presenting cells prepared by the preparation method of the present invention.
The antigen-presenting cells produced by the method for preparing antigen-presenting cells of the present invention can be used as a pharmaceutical composition in combination with a pharmaceutically acceptable solvent (e.g., physiological saline).
The method of use of the pharmaceutical composition comprises: intravenous injection, subcutaneous injection, intradermal injection, etc., drip injection, and injection from an artery near a lesion.
Further, the antigen presenting cells are derived from the patient himself.
Antigen-presenting cells are derived from cells produced by the patient themselves, and exert their effects without being rejected by the immune system in the patient.
Antigen-presenting cells administered into the body migrate to lymph nodes and the like, and stimulate healthy T cells including antigen-specific Th1 cells, killer T cells, and the like. The activated antigen-specific Th1 cells produce IL-2 and IFN-gamma, enhancing cellular immunity.
Further, the pharmaceutical composition activates Th1 cells in the patient.
Further, an antigen-presenting cell active agent containing a nucleotide as an active ingredient.
The antigen-presenting cell activator of the present invention is an antigen-presenting cell activator containing a nucleotide as an active ingredient.
An antigen presenting cell active agent is an agent that enhances the function of an antigen presenting cell when contacted with the antigen presenting cell in vivo or in vitro.
The antigen-presenting cell active agent of the present invention comprises nucleotides as an active ingredient to induce an immune response function of antigen-presenting cells and to enhance Th1 cells (C4) specifically recognized by absorbed antigens+T cells).
The antigen-presenting cell active agent of the present invention can be prepared by adding nucleotides to a physiological solvent, such as PBS and physiological saline.
The antigen-presenting cell activator of the present invention may be used in vitro at a final concentration of 10. mu.M to 1000. mu.M.
The method for exerting the effect in vivo includes a method of injecting the antigen-presenting cell activator of the present invention comprising saline and nucleotides into the body by injection, intravenous injection, subcutaneous injection, intradermal injection, lymph node injection, etc., or a method of intravenous drip injection, or a method of injecting the antigen-presenting cell activator into the body by arterial injection in the vicinity of a lesion.
By administering the antigen-presenting cell activator of the present invention to a patient, the antigen-presenting cells of the patient are activated, and the activated antigen-presenting cells cause Th1 cells (CD 4) in the patient+T cells) to improve the condition.
Preferably, the nucleotide is a ribonucleotide.
Preferably, the nucleotide is a nucleoside-phosphate.
Preferably, the nucleotide is uridylic acid or adenylic acid.
As can be seen from the above-described technical solutions, the present disclosure provides a method for preparing antigen-presenting cells, which can activate antigen-specific CD4, as compared with the prior art+T cells. By utilizing the properties of the antigen presenting cells, a medicament for treating cancer and infectious diseases can be produced; in addition, since the nucleotide has the property of activating antigen-presenting cells, it can be used as an antigen-presenting cell active agent; antigen presenting cells can be modulated in vitro by using antigen presenting cell active agents; if administered to a patient, the condition of the patient can be improved by activating antigen presenting cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing of the CD4 of the present invention+Production of IFN- γ in T cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Sample preparation of dendritic cell precursor cells: peripheral blood, bone marrow fluid, umbilical cord blood and the like are provided, and the peripheral blood is preferably used in view of availability and reduction in physical burden on a patient. The amount of peripheral blood collected is appropriately set based on the extent of not causing a physical burden on the provider. The blood collecting method comprises collecting whole blood with vacuum blood collecting tube and blood collecting back, adding heparin phosphate and citric acid into the collected blood, and anticoagulating.
In addition, when a large amount of cells need to be obtained, monocytes may be collected by the component blood collection device. Isolating monocytes, including dendritic cell precursors, from the collected sample. Separation methods, any method of separating monocytes from red blood cells may be used; for example, the Fico-Pacoe density gradient method is generally used; in addition, in order to remove platelets and the like from the separated monocytes, the cells are washed several times with a medium, physiological saline, phosphate buffered physiological saline (PBS), or the like.
Separating monocytes (CD 14-positive cells) that are precursors of dendritic cells from the separated monocytes; monocytes can be recovered by magnetic bead sorting of the cells.
Transferring the separated mononuclear cells to a culture vessel, and culturing at 34-38 ℃ with 2-10% CO2When cultured for 1 hour or more under the conditions, the cells adhere to the bottom surface (wall surface) of the culture flask, and the adhered cells can also be used as precursor cells of dendritic cells.
The obtained precursor cells are stimulated with cytokine or the like to differentiate immature dendritic cells and mature dendritic cells. As a medium for culturing, commercially available cell culture media such as AIM-V medium, RPMI-1640 medium, Dulbecco's Modified Eagle Medium (DMEM), TIL (immune biology research institute, K.K.), epidermal keratinocyte medium (KBM, Seiko Bio Inc.), and Iskov medium (IMEM) were used. Further, as necessary, 0.5% to 20% of bovine serum, bovine fetal serum (hereinafter referred to as FBS), human serum, human blood color, or the like may be added.
In order to obtain immature dendritic cells, dendritic cell precursor cells in a medium to which differentiation-inducing factors are added may be cultured.The factors for differentiation induction of immature dendritic cells include granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 4(IL-4), Stem Cell Factor (SCF) IL-13, tumor necrosis factor α (TNF- α), and optionally IL-1, IL-2, and IL-3, and the culture conditions for differentiation induction are 34-38 deg.C, 37 deg.C, and 2-10% CO2More preferably, it is at least 5% CO2Culturing for 5-7 days under the condition.
In order to obtain mature dendritic cells, the obtained immature dendritic cells are cultured in a medium containing differentiation-inducing factors, such as GM-CSF, IL-4, SCF, IL-1 β, IL-6, IL-13, TNF- α, and prostaglandin E2(PGE2), and IL-1, IL-2, and IL-3 may be added as necessary, and for culturing and maturation, the cells may be cultured at 34-38 deg.C, 37 deg.C, 2-10% CO2More preferably, it is at least 5% CO2Culturing for 2-3 days under the condition.
Hematopoietic stem cells (CD 34-positive cells) which are dendritic cell precursors are prepared, and one or a combination of more of GM-CSF, TFN- α, ft-3 ligand, c-kit ligand, and thrombopoietin is added to a culture medium and cultured to obtain dendritic cells.
Example 2
The nucleotide-assisting effect in the presentation of the solubilized antigen protein was confirmed by the following procedure.
< preparation of mouse rTRP-2 >
Recombinant mouse TRP-C (rTRP-2) was used for the experiments. rTRP-2 is a cDNA encoded between glutamic acid (E) at residue 56 and serine (S) at residue 472 based on the amino acid arrangement described in ACS.P220812 of CROISS-PROT database. The isolated cDNA was inserted into plasmid pET19b/mdT, and E.coli Rosetta-gami2(DE3) pLysS was transformed with pET19b/mdT to give a recombinant protein having His-tag at the N-terminus. This protein forms inclusion bodies and is insoluble.
< modulation of DC >
Male mice, C57BL/6, were purchased for 8 weeks of age, raised in an SPF environment and bone marrow cells were collected after week 9. The collected bone marrow cells were inoculated in AIM-V medium containing 0.2% mouse serum, to which 10ng/ml mouse IL-4 and 20ng/ml mouse GM-CSF were added, and at 37 ℃ with 5% CO2The next day of culture, the next day, the same fresh medium was changed. The floating cells were centrifuged and recovered at medium exchange and returned to the flask. The medium was changed every day, and the planktonic cells were recovered on the ninth day of bone marrow cell culture. Suspended in fresh medium, and cultured with 1. mu.g/ml Lipopolysaccharide (LPS) for one day, and the floating cells were used as mature DCs.
< preparation of DC and immunization of mouse >
Mouse rTRP-2 (insoluble) was added to UMP or guanosine (hereinafter referred to as GMP) and solubilized in PBS containing 10% glycerol. Adding a sample containing solubilized mouse rTRP-2 to mature DC (mDC) and introducing by electroporation; nucleotides were added to the protein solution, and the culture was continued overnight after the electroporation treatment, and mice were immunized. The abdominal cavity of C57BL/6 mouse (n-2) was filled with 1x106One DC, once a week, counted three times. Splenocytes were collected from each mouse 6 days after immunization.
< Mixed lymphocyte reaction >
From harvested splenocytes, CD4 was used+TCell Isolation Kit, CD4 by negative selection+T cells. Introduction of solubilized rT-2 protein into DC as Antigen Presenting Cell (APC) for CD4 transformation+T cells as a Mixed Lymphocyte Reaction (MLR) experiment of the resolder. APC 3X 105Of (1, CD 4)+T cells 3X 106After 3 days of culture in 6mL of AIM medium, 10U/mL of IL-2 was added and the culture was continued for one more day. After the culture, the supernatant was collected by centrifugation at 3000g at 4 ℃ for 5min, and the IFN-. gamma.concentration in the supernatant was quantified using a MousIFN-ELISA element (PIERASICE, EM1001), and the results are shown in FIG. 1.
The data in figure 1 shows the mean (error bars are SD) of 2 mice per group. Only DCs without added antigen (mDC) and NormalMurine CD4+T cells (CD 4)+) No IFN-gamma was detected. DCs obtained by pulsing TRP-2 peptide (type I restraint) and CD4 derived from immunized mice+Amount of IFN-. gamma.produced by MLR (peptide) of T cells, and DC without antigen addition and CD 4-derived mice immunized therewith+MLR (Co- (-)) of T cells, DCs subjected to electroporation (Ep) without addition of antigen, and CD4 of immunized mice+The amount of IFN-. gamma.produced by MLR (Ep- (-) of T cells was essentially the same. On the other hand, the solubilized TRP-2 protein to which UMP-2 was added was introduced into DCs by electroporation, and MLR (Co-T (U)) derived from CD 4+ T cells in the DCs and the immunized mice increased IFN-. gamma.production efficiently (P. sub.0.017) as compared with Co- (-). Introduction of UMP-2-added solubilized rTRP-2 protein into DCs by electroporation, and CD4 of immunized mice+MLR (Ep-T) (Ep-T (u)) of T cells was further expanded, and IFN- γ production was more efficiently increased (P ═ 0.045) than that of co-culture. The results show that electroporation produces more IFN-. gamma.than co-culture. In addition, the soluble TRP-2 protein to which GMP was added was introduced into DC by electroporation, and the DC and CD4 of the mouse immunized with the DC+T cells have MLR (Ep-T (G)) and Ep- (U) that produce more IFN-. gamma.than does T.
In conclusion, under in vitro conditions in the presence of nucleotides, antigen-specific Th1 cells were able to recognize the added antigen, and antigen-presenting cells to which the antigen was added had a stimulatory effect on Th1 cells.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A method for preparing antigen-presenting cells, comprising adding an antigen to antigen-presenting cells under in vitro conditions in the presence of nucleotides; the antigen-added antigen presenting cells are dendritic cells.
2. The method for preparing antigen-presenting cells according to claim 1, wherein the nucleotides are ribonucleotides.
3. The method for preparing antigen-presenting cells according to claim 1, wherein the nucleotide is a nucleoside-phosphate.
4. The method for preparing antigen presenting cells according to claim 1, wherein the nucleotide is uridylic acid or adenylic acid.
5. The method for preparing antigen-presenting cells according to claim 1, wherein the antigen-specific Th1 cells recognize the added antigen, and the antigen-presenting cells stimulate Th1 cells.
6. An antigen-presenting cell prepared by the method for preparing an antigen-presenting cell according to any one of claims 1 to 5.
7. An antigen-presenting cell active agent containing a nucleotide as an active ingredient.
8. The antigen-presenting cell active agent comprising a nucleotide as an active ingredient according to claim 7, wherein the nucleotide is a ribonucleotide.
9. The antigen-presenting cell active agent comprising a nucleotide as an active ingredient according to claim 7, wherein the nucleotide is a nucleoside-phosphate.
10. The antigen-presenting cell active agent comprising a nucleotide as an active ingredient according to claim 7, wherein the nucleotide is uridylic acid or adenylic acid.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004015056A2 (en) * | 2002-08-12 | 2004-02-19 | The University Of Queensland | Immunomodulating compositions, processes for their production and uses therefor |
| CN102459308A (en) * | 2009-05-01 | 2012-05-16 | 迈世耐特股份公司 | Method for solubilizing insoluble protein and/or peptide |
| JP2012107007A (en) * | 2010-10-28 | 2012-06-07 | Medeinetto:Kk | Method for preparation of antigen-presenting cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004015056A2 (en) * | 2002-08-12 | 2004-02-19 | The University Of Queensland | Immunomodulating compositions, processes for their production and uses therefor |
| CN102459308A (en) * | 2009-05-01 | 2012-05-16 | 迈世耐特股份公司 | Method for solubilizing insoluble protein and/or peptide |
| JP2012107007A (en) * | 2010-10-28 | 2012-06-07 | Medeinetto:Kk | Method for preparation of antigen-presenting cell |
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